Proteins specific Solubility

The study of protein function as it applies to covalent and noncovalent molecular changes associated with specific binding is of fundamental interest to those involved with the discovery of therapeutic proteins. Discovering soluble receptors that bind the cytokines that elicit inflammatory immune responses is one of many... 

Figure 7. Variation of arogenate dehydrogenase levels as a function of the physiological phase of growth in suspension cultures of Nicotiana sil-vestris. A stationary-phase inoculum was diluted into fresh medium and followed throughout the lag (L), exponential (E), and stationary (S) phases of growth. The hatched bar indicates the activity levels of EE cells, i.e., cells maintained continuously in exponential growth for 10 or more generations (53). Profiles are shown in which activity is related to soluble protein (specific activity), to cell number, or to dry weight.

Even if relatively new, HF FIFFF has been used to separate supramicrometer particles, proteins, water-soluble polymers, and synthetic organic-soluble polymers. Particle separation in HF FIFFF has recently been improved, reaching the level of efficiency normally achieved by conventional, rectangular FIFFF channels. With these channel-optimized HF FIFFF systems, separation speed and the resolution of nanosized particles have been increased. HF FIFFF has recently been examined as a means for off-line and on-line protein characterization by using the mass spectrometry (MS) through matrix-assisted laser desorption ionization time-of-flight mass spectrometry (M ALDl-TOF MS) and electrospray ionization (ESl)-TOF MS, as specific detectors. On-line HF FIFFF and ESl-TOF MS analysis has demonstrated the viability of fractionating proteins by HF FIFFF followed by direct analysis of the protein ions in MS [38]. 

Serial affinity chromatography (SAC) method, described in Subheading 3.4 in detail, was used for identification of the specific binding protein since solubility of Valdecoxib was too low to per-fome the traditional competition method. 

Traditionally, milk was paid for mainly on the basis of its fat content but milk payments are now usually based on the content of fat plus protein. Specifications for many dairy products include a value for protein content. Changes in protein characteristics, e.g. insolubility as a result of heat denaturation in milk powders or the increasing solubility of cheese proteins during ripening, are industrially important features of these products. 

Inositol trisphosphate, a water-soluble compound, diffuses from the plasma membrane to the endoplasmic reticulum, where it binds to specific IP3 receptors and causes Ca2+ channels within the ER to open. Sequestered Ca2+ is thus released into the cytosol (step (5)), and the cytosolic [Ca2+] rises sharply to about 10 6 m. One effect of elevated [Ca2+] is the activation of protein kinase C (PKC). Diacylglycerol cooperates with Ca2+ in activating PKC, thus also acting as a second messenger (step (6)). PKC phosphorylates Ser or Thr residues of specific target proteins, changing their catalytic activities (step (7)). There are a number of isozymes of PKC, each with a characteristic tissue distribution, target protein specificity, and role. 

At every step in these processes vesicles are formed and are carried to the next destination where the vesicles fuse with the new membrane and discharge their contents.80-853 This remarkable process is complex and highly specific. Rothman proposed that the vesicles to be transported are "docked" on appropriate receptor molecules (called SNARES) on the destination membrane. This is accomplished with the aid of specific soluble marker proteins (called SNAPS) with surfaces complementary to those of the receptors.80 Proteins are sorted in this way according to their destination signals. The Golgi system is considered further in Chapter 20. 

Before selecting a method to measure a specific aspect of protein functionality, one must decide on the complexity of the testing matrix. Researchers have used a single purified protein, a crude extract of proteins, a prototype food product, or an actual product to study protein functionality. For meat studies, formulated meat systems, ground muscle, myofibrillar proteins, salt-soluble proteins, actomyosin,... 

The process whereby newly synthesized proteins are directed to specific locations is referred to as protein targeting. Soluble lysosomal hydrolases are targeted to lysosomes... 

Additional application of IAP has been in the production of species-specific antibodies. Martin et al (136) first purified crude animal protein extracts by IAP. These purified proteins were then used to raise horse-specific monoclonal antibodies. This approach helped in significantly shortening the isolation time of species-specific monoclonal antibodies. A similar approach was used for pig-specific soluble muscle protein polyclonal antibodies (137). 

In meat emulsions, a specific soluble fraction of proteins was shown to function as the key emulsifier (22). However, Lin et al. (15) showed that sunflower meal was superior to soybean and sunflower protein concentrates or isolates in emulsification capacity. These authors suggest that nonprotein constituents of seeds may contribute to the formation of emulsions and aid in the formation of whipped foams. 

Because they are hydrophobic molecules, estrogens easily diffuse across cell membranes. When inside a cell, estrogens bind to highly specific, soluble receptor proteins. Estrogen receptors are members of a large family of proteins that act as receptors for a wide range of hydrophobic molecules, including other steroid hormones, thyroid hormones, and retinoids. 

Shinohara, N., Huang, Y-Y. and Maroyama, A. (1991). Specific suppression of antibody responses by soluble protein-specific, class II restricted cytotoxic T lymphocyte clones. Eur. J. Immunol. 21, 23-27. 

Many proteins are easily resolubilised in a small amount of the buffer to be used in the next chromatographic step. However, an agent, selected from Table 13, may be required for less soluble proteins. Specific conditions will depend upon the specific protein. These agents must always be removed to allow complete re-folding of the protein and maximise recovery of mass and activity. 

SR-proteins can be expressed as recombinant proteins in Escherichia coli, but they lack the post-translational phosphorylation of the serine residues and are poorly soluble in the absence of chaotropic reagents. It is possible to phosphorylate bacterially produced protein by preincubation in nuclear extract or, even more efficiently, by the addition of purified recombinant SR-protein specific kinases Clk/Sty8 or SRPK.9 Soluble and phosporylated SR-proteins can also be produced in insect cells using the baculovirus system10 although it is not resolved whether these proteins behave exactly as those produced in human cells. 

The conversion of famesyl pyrophosphate to squalene marks the transition from water-soluble to water-insoluble intermediates in the biosynthesis of cholesterol. When the effect of liver cytosol or SCPj on the conversion of famesyl pyrophosphate to squalene was investigated, neither rat liver cytosol nor partially purified SCPj had any significant effect on this conversion [23]. Therefore, microsomal squalene synthetase performs its catalytic function without responding to the mediating effect of any specific soluble protein. 

Two types of soluble binding protein have been described, nonspecific serum proteins and cytokine-specific soluble receptor proteins. The major nonspecific serum protein capable of binding cytokines appears to be a2-macroglobuIin (B57, B58, Mil). A number of soluble cytokine inhibitors related to the relevant receptor have been described. The soluble form of the IL-2R a chain has been found in the serum of apparently normal individuals and is increased in the serum of individuals with inflammatory diseases such as rheumatoid arthritis (W31). Similar molecules have been described for IL-1, IL-6, IFNy, and IL-7 (F7, N14, S62). The mechanism of release has not been properly established but appears to require proteolytic cleavage of the membrane-bound receptor. Soluble inhibitors for two other cytokines, IL-4 and TNF, appear to be derived by alternative RNA splicing sites that give rise to receptors lacking a transmembrane sequence and that are secreted (M50, S22). 

---